The present invention relates to the isolation of a novel Pseudomonas species and identification of novel heterocyclic dioxygenases derived from this organism. The heterocyclic dioxygenase described herein is useful in the production of indigo in recombinant organisms.
Pseudomonads are a diverse group of organisms capable of mineralizing biotic (e.g., camphor), xenobiotic (various chlorophenyl and biphenyl compounds) and fossil organic molecules (such as toluene or naphthalene). These capabilities are usually encoded by groups of genes collected in operons on extrachromosomal elements. A number of different enzyme classes are involved in the initial oxidation of these compounds. For example, camphor is degraded by a P450 monooxygenase while toluene, chlorobiphenyl and biphenyl, and naphthalene are oxidized by aromatic dioxygenases.
In the era of molecular biology, it was discovered that when certain aromatic dioxygenases were cloned into Escherichia coli and these were subsequently grown on a rich medium, colonies turned blue. It was later determined that the blue color was due to the conversion of tryptophan in the medium to indole by E. coli tryptophanase and its subsequent oxidation by the aromatic dioxygenases to indolediol, which in turn, in the presence of molecular oxygen, spontaneously further oxidized to indigo, leading to the observed blue coloration. Dioxygenases whose primary substrates are heterocyclic compounds, particularly where the substrate is indole, have not previously been characterized in detail. The heterocyclic dioxygenase reported here is capable of indole oxidation, leading to indigo formation.
The blue dye indigo is one of the oldest dyestuffs known to man. Its use as a textile dye dates back to at least 2000 BC. Until the late 1800s indigo, or indigotin, was principally obtained from plants of the genus Indigofera, which range widely in Africa, Asia, the East Indies and South America. As the industrial revolution swept through Europe and North America in the 1800s, demand for the dye""s brilliant blue color led to its development as one of the main articles of trade between Europe and the Far East. In 1883, Adolph von Baeyer identified the formula of indigo: C16H10N2O2. In 1887, the first commercial chemical manufacturing process for indigo was developed. This process, still in use today, involves the fusion of sodium phenylglycinate in a mixture of caustic soda and sodamide to produce indoxyl. The process"" final product, indoxyl, oxidized spontaneously to indigo by exposure to air.
Current commercial chemical processes for manufacturing indigo result in the generation of significant quantities of toxic waste products. Obviously, a method whereby indigo may be produced without the generation of toxic by-products is desirable. One such method which results in less toxic by-product generation involves indigo biosynthesis by microorganisms.
Ensley et al. ((1983) Science 222:167-169) found that a DNA fragment from a transmissible plasmid isolated from the soil bacterium Pseudomonas putida enabled Escherichia coli that had been stably transformed with a plasmid harboring the fragment to synthesize indigo in the presence of indole or tryptophan. Ensley et al. postulated that indole, added either as a media supplement or produced as a result of enzymatic tryptophan catabolism, was converted to cis-indole-2,3-dihydrodiol by the previously identified multi-component-enzyme naphthalene dioxygenase (NDO) encoded by the P. putida DNA fragment. The indole-2,3-dihydrodiol so produced spontaneously lost water forming indoxyl and was then oxidized to indigo upon exposure to air.
NDO had previously been found to catalyze the oxidation of the aromatic hydrocarbon naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene (Ensley et al. (1982) J. Bacteriol. 149:948-954). U.S. Pat. No. 4,520,103, incorporated by reference, describes the microbial production of indigo from indole by an aromatic dioxygenase enzyme such as NDO. The NDO enzyme is comprised of multiple components: a reductase polypeptide (Rd, molecular weight of approximately 37,000 daltons (37 kD)); an iron-sulfur ferredoxin polypeptide (Fd, molecular weight of approximately 13 kD); and a terminal oxygenase iron-sulfur protein (ISP). ISP itself is comprised of four subunits having an xcex12xcex22 subunit structure (approximate subunit molecular weights: xcex1, 55 kD; xcex2, 21 kD). ISP is known to bind naphthalene, and in the presence of NADH, Rd, Fd and oxygen, to oxidize it to cis-naphthalene-dihydrodiol. Fd is believed to be the rate-limiting polypeptide in this naphthalene oxidation catalysis, (see U.S. Pat. No. 5,173,425, incorporated herein by reference, for a thorough discussion of the various NDO subunits and ways to improve them for purposes of indigo biosynthesis).
In addition, aromatic dioxygenases other than NDO may also be useful in the biosynthetic production of indigo, for example, a toluene monooxygenase (TMO) such as that from Pseudomonas (P. mendocina) capable of degrading toluene was also able to produce indigo when the culture medium was supplemented with indole. For details, see U.S. Pat. No. 5,017,495, incorporated herein by reference. In principle, any enzyme capable of introducing a hydroxyl moiety into the 3-position of indole to give indoxyl is a candidate for use in the biosynthetic production of indigo. This includes single component flavin containing monooxygenases.
Most, if not all, oxygenases described in the art for use in oxidation of the substrate indole, as for example in the production of indigo, are aromatic oxygenases. While these enzymes have been successfully employed in the synthesis of indigo, there is a need for an enzyme or class of enzymes which have as a primary substrate heterocyclic compounds such as indole. Such heterocyclic oxygenases are believed to be advantageous over aromatic oxygenases in oxidizing indole and ultimately in indigo production.
The following terms will be understood as defined herein unless otherwise stated. Such definitions include without recitation those meanings associated with these terms known to those skilled in the art.
Tryptophan pathway genes useful in securing biosynthetic indole accumulation include a trp operon, isolated from a microorganism as a purified DNA molecule that encodes an enzymatic pathway capable of directing the biosynthesis of L-tryptophan from chorismic acid. (A. J. Pittard (1987) Biosvnthesis of Aromatic Amino Acids in Escherichia coli and Salmonella typhimurium, F. C. Neidhardt, ed., American Society for Microbiology, publisher, pp. 368-394.) Indole accumulation is enabled by modification of one or more of the pathway""s structural elements and/or regulatory regions. This modified trp operon may then be introduced into a suitable host such as a microorganism, plant tissue culture system or other suitable expression system. It should be noted that the term xe2x80x9cindole accumulationxe2x80x9d does not necessarily indicate that indole actually accumulates intracellularly. Instead, this term can indicate that there is an increased flux of carbon to indole and indole is made available as a substrate for intracellular catalytic reactions such as indoxyl formation and other than the formation of L-tryptophan. In the context of this invention, the xe2x80x9caccumulatedxe2x80x9d indole may be consumed in the conversion of indole to indoxyl by an oxygenase such as the aromatic dioxygenase NDO, or an aromatic monooxygenase such as TMO, or it may actually build up intracellularly and extracellularly, as would be the case when the desired end product is indole or one of its derivatives.
A suitable host microorganism or host cell is an autonomous single-celled organism useful for microbial indole and/or indigo production and includes both eucaryotic and procaryotic microorganisms. Such host microorganism contains all DNA, either endogenous or exogenous, required for the production of indole, indoxyl and/or indigo, from glucose, as a bioconversion from tryptophan, or, in the case of producing indoxyl or indigo, from indole. Useful eucaryotes include organisms like yeast and fungi or plants. Prokaryotes useful in the present invention include, but are not limited to, bacteria such as E. coli, P. putida and Salmonella typhimurium. 
Biosynthetic conversion of indole to indigo is meant to include indoxyl oxidation to indigo mediated by molecular oxygen or air.
A DNA fragment, as used herein, may encode regulatory and/or structural genetic information. A DNA fragment useful in the present invention shall also include: nucleic acid molecules encoding sequences complementary to those provided; nucleic acid molecules (DNA or RNA) which hybridize under stringent conditions to those molecules that are provided; or those nucleic acid molecules that, but for the degeneracy of the genetic code, would hybridize to the molecules provided or their complementary strands. xe2x80x9cStringentxe2x80x9d hybridization conditions are those that minimize formation of double stranded nucleic acid hybrids from non-complementary or mismatched single stranded nucleic acids. In addition, hybridization stringency may be affected by the various components of the hybridization reaction, including salt concentration, the presence or absence of formamide, the nucleotide composition of the nucleic acid molecules, etc. The nucleic acid molecules useful in the present invention may be either naturally or synthetically derived.
A xe2x80x9cheterologous or exogenousxe2x80x9d DNA fragment is one that has been introduced into the host microorganism by any process such as transformation, transfection, transduction, conjugation, electroporation, etc. Additionally, it should be noted that it is possible that the host cell into which the xe2x80x9cexogenousxe2x80x9d DNA fragment has been inserted may itself also naturally harbor molecules encoding the same or similar sequences. For example, when E coli is used in this invention as the host strain, it is recognized that, normally, the host naturally contains, on its chromosome, a trp operon capable of directing the synthesis of L-tryptophan from chorismic acid under conditions enabling trp operon expression. A molecule such as this is referred to as an xe2x80x9cendogenousxe2x80x9d DNA molecule.
A stably transformed microorganism is one that has had one or more exogenous DNA fragments introduced such that the introduced molecules are maintained, replicated and segregated in a growing culture. Stable transformation may be due to multiple or single chromosomal integration(s) or by extrachromosomal element(s) such as a plasmid vector(s). A plasmid vector is capable of directing the expression of polypeptides encoded by particular DNA fragments. Expression may be constitutive or regulated by inducible (or repressible) promoters that enable high levels of transcription of functionally associated DNA fragments encoding specific polypeptides such as the structural genes of a trp operon modified as described herein.
An xe2x80x9cisatin-removing enzyme,xe2x80x9d as used herein, is any enzyme which comprises activity resulting in the inhibition, removal, inactivation, degradation, hydrolysis or binding (sequestering) of isatin, whether such enzyme causes the formation of isatic acid or any other degradation product. A preferred isatin-removing enzyme useful in the present invention is an isatin hydrolase such as the hydrolase isolated from Pseudomonas putida (WW2) herein, deposited in accordance with the Budapest Treaty on International Recognition of the Deposits of Microorganisms for the Purpose of Patent Procedures as Deposit ATCC #55763.
Regardless of the exact mechanism utilized for expression of enzymes necessary for the microbial production of indole, indoxyl and/or indigo, it is contemplated that such expression will typically be effected or mediated by the transfer of recombinant genetic elements into the host cell. Genetic elements as herein defined include nucleic acids (generally DNA or RNA) having expressible coding sequences for products such as proteins, specifically enzymes, apoproteins or antisense RNA, which express or regulate expression of relevant enzymes (i.e., isatin hydrolase, tryptophan synthase, NDO, etc.). The expressed proteins can function as enzymes, repress or derepress enzyme activity or control expression of enzymes. Recombinant DNA encoding these expressible sequences can be either chromosomal (integrated into the host cell chromosome by, for example, homologous recombination) or extrachromosomal (for example, carried by one or more plasmids, cosmids and other vectors capable of effecting the targeted transformation). It is understood that the recombinant DNA utilized for transforming the host cell in accordance with this invention can include, in addition to structural genes and transcription factors, expression control sequences, including promoters, repressors and enhancers, that act to control expression or derepression of coding sequences for proteins, apoproteins or antisense RNA. For example, such control sequences can be inserted into wild-type host cells to promote overexpression of selected enzymes already encoded in the host cell genome, or alternatively they can be used to control synthesis of extrachromosomally encoded enzymes.
The recombinant DNA can be introduced into the host cell by any means, including, but not limited to, plasmids, cosmids, phages, yeast artificial chromosomes or other vectors that mediate transfer of genetic elements into a host cell. These vectors can include an origin of replication, along with cis-acting control elements that control replication of the vector and the genetic elements carried by the vector. Selectable markers can be present on the vector to aid in the identification of host cells into which genetic elements have been introduced. Exemplary of such selectable markers are genes that confer resistance to particular antibiotics such as tetracycline, ampicillin, chloramphenicol, kanamycin or neomycin.
A means for introducing genetic elements into a host cell utilizes an extrachromosomal multi-copy plasmid vector into which genetic elements in accordance with the present invention have been inserted. Plasmid borne introduction of the genetic element into host cells involves an initial cleaving of a plasmid vector with a restriction enzyme, followed by ligation of the plasmid and genetic elements encoding for the targeted enzyme species in accordance with the invention. Upon recircularization of the ligated recombinant plasmid, infection (e.g., packaging in phage lambda) or other mechanism for plasmid transfer (e.g., electroporation, microinjection, etc.) is utilized to transfer the plasmid into the host cell. Plasmids suitable for insertion of genetic elements into the host cell are well known to the skilled artisan.
The present invention provides a novel microorganism of the genus Pseudomonas, the species of which is preliminarily identified as a putida, and strain designation WW2. The designation therefore is Pseudomonas putida strain WW2. This strain has been deposited at the American Type Culture Collection, Rockville, Md. as strain ATCC #55763.
Also provided is an enzyme isolated from P. putida WW2 which is an indole oxidase, as well as methods for producing the oxidase and compositions of matter comprising the oxidase as a single component lndole is the primary substrate for this enzyme expressed from WW2 since the organism can grow on indole as the sole substrate providing carbon, nitrogen and energy. This enzyme is hereafter referred to as an indole oxidase or dioxygenase and has the following physio-chemical properties: (a) enzyme action: catalyzes in presence of NADH or NADPH and FAD as a cofactor, the oxidation of indole to indoxyl or indolediol; and (b) pH optimum of from about 7-9, preferably 8.0.
A further aspect of this invention is the use of the indole oxidase or dioxygenase to oxidize indole toga precursor which spontaneously converts to indigo. Thus, there is provided an improved method for the biocatalytic production of indigo in a suitable host-microorganism. Suitable host microorganisms include but are not limited to host organism(s) expressing (either exogenously or endogenously) indole oxygenase activity and/or isatin hydrolase activity. Such organisms are cultivated under conditions facilitating the expression of the indole oxidase activity and/or isatin hydrolase. By way of example, a suitable microorganism could be P. putida ATCC #55763 which expresses endogenously both indole oxidase and isatin hydrolase activity. This embodiment preferably would include a modification of ATCC #55763 to block conversion of indole to compounds along its indole degradation pathway to compounds other than indigo. Similarly, a suitable host organism could express endogenously or exogenously any one or more of the enzymatic activities necessary to convert glucose, tryptophan (tryptophanase) or indole (indole oxidase) to indigo. As such, a suitable host organism could be a procaryote or eucaryote transformed or transfected with DNA encoding one or more of the following: shikimic acid pathway enzymes, indole oxidase, a tryptophan operon (or modified operon) and/or isatin hydrolase.
The oxidase enzyme of the present invention is a multicomponent enzyme that can utilize nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine phosphate dinucleotide (NADPH), requires flavin adenine dinucleotide (FAD) and its activity is stimulated by the presence of iron in a cell-free extract.
The enzyme has very low activity with naphthalene, indicating significant differences between the present enzyme and naphthalene dioxygenase previously reported in the production of indigo in E coli. 
Another aspect of the present invention is the cloning and sequencing of the gene encoding a preferred oxidase enzyme, indole oxidase.